Process of extracting small molecular ingredients from biological materials under super high pressure

ABSTRACT

The process of extracting small molecular ingredients from biological materials under super high pressure is a process which makes use of super high pressure to extract small molecular active ingredients from biological materials, especially natural products, which mainly consists of extracting the mixture of solid raw material and extraction solvent under increased pressure. Said process comprises the following steps: the step of pretreatment, crash and formulation; the step of closure, charging the formulated mixture of raw material and extraction solvent into high pressure container, then closing the high pressure container; the step of increasing pressure, increasing the pressure of the high pressure container from 100 Mpa to the predefined pressure of 1000 Mpa; the step of holding pressure, holding the predefined pressure for 3-30 minutes; the step of releasing pressure, releasing the pressure of the high pressure container to normal pressure, removing the mixture. The present invention modifies the traditional extraction process of small molecular ingredients in biological materials, which not only makes extracts avoiding heating, but also has the advantages of high extraction yields, rapid extraction and broad extraction solvents.

The present application is the U.S. National Phase of PCT InternationalApplication No. PCT/CN02/00929 filed Dec. 30, 2002 which claims thebenefit of Chinese Patent Application No. 01138981.8 filed Dec. 30,2001.

FIELD OF THE INVENTION

The present invention relates to a process of extracting from biologicalmaterials under super high pressure, especially to a process ofextracting small molecular active ingredients from biological materialsunder super high pressure.

The biological materials include the total, partial, tissue, partialtissue or other parts of plants, animals and microorganisms.

The small molecules include the molecules that comprise fewer atoms withlower molecular weight. Generally, the molecular weight is lower than10,000.

The active ingredients include the components that can be used fortreatment of diseases, physiological accommodation, improvement ofhealth, promotion of growth, as well as improvement of color, flavourand taste of food, etc. Natural produces comprise many kinds of activeingredients, such as alkaloids, glucosides, organic acids, volatileoils, terpenoids, flavonoids, steroids, oligosaccharides,polysaccharides, coumarins, lignins, saponin, amino acids, peptides andproteins, enzymes, tannins, resin, pigments of plant, oils, wax,inorganic salts, etc. These active ingredients are generally dividedinto hydrophilic and lipophilic substance according to their solubility.Many ingredients will be inactive or active lowered after heating.

The super high pressure, said in the invention, is hydrostatic pressureof 100 MPa˜1000 MPa.

BACKGROUND OF THE INVENTION

There are many methods of extracting active ingredients frombiomaterials. The methods for extracting active ingredients from naturalproducts are solvent exaction (including immersion, leaching, refluxing,boiling), vapour distillation and sublimation, Gao Yuanjun, Thedevelopment and processing of the wild plants in China, Chinese lightindustry press, Beijing, 1997.February. The methods to extract naturalcomponents are the solvent technology, vapour distillation, andsublimation. The latter two are limitedly used, the solvent technologyis used in most conditions, Yao xinsheng, Chemistry of natural drugs,People's Medical Publishing House, Beijing, 1998.June. These methodshave many advantages, for example, many kinds of solvent can be used,many active ingredients can be obtained and the extraction yield ishigher. However, there are also many shortages, for example, difficultyof separating and purifying, largely denaturing of the activeingredients, much loss of the ingredient by volatilization, and longextraction time, etc. Although several technologies have been developedrecently, such as enzyme technology, ultra-micro-pulverizing technologyetc., the above shortages are not be eradicated.

The newest technology supercritical CO₂ extraction has solved some ofthe above problems, particularly has a notable advantage of separationand purification, and accordingly has become a very important extractiontechnology. However, Zhu Zhiqiang described in Supercritical fluidtechnology, principle and application, Chemical industry press, Beijing,2000.March: CO₂ has lipophilic property. The fragrant oils, fat,alcohol, aldehyde, ketone, wax, the light fraction of resin can beselectively extracted by supercritical CO₂ , —saccharides and salts areinsoluble in supercritical CO₂. The solubility of alkaloids,polysaccharides, steroids, terpenoids, glycosides, saponin orflavonoids, etc. in supercritical CO₂ is very poor, which makes itdifficult for these substances to be extracted by supercritical CO₂exaction. Additionally, alkaloids, nicotine and caffeine may react withother components, such as citric acid and caffetannic acid, to formsalts, which causes it difficult to extract these ingredients bysupercritical CO₂. In addition, there are other disadvantages ofsupercritical CO₂ extraction, for example, expensive equipment and highcost of operation.

DESCRIPTION OF THE INVENTION

This invention provides a new process of extracting small molecularingredients from biomaterials under super high pressure with highextraction yield and short extraction time to overcome the disadvantagesof present extraction methods.

The process of extracting small molecular ingredients from biomaterialsaccording to the invention is a process of extracting small molecularingredients form the mixture of the solid raw biomaterial and solventunder super high pressure. The procedures are as follows:

The step of pretreatment, crashing and formulation: the solid rawbiomaterial is pretreated first, including roguing, cleaning, ormacerating, and then crashed. Finally mix the crashed biomaterials withproper solvent by the proper ratio homogeneously.

The step of closure: put the above mixture into the high pressurecontainer firstly and then close the container. Alternatively, close thecontainer firstly and then charge the mixture into the container.

The step of increasing pressure: The pressure of the pressure containeris increased from normal pressure to the predefined pressure of 100MPa˜1000 MPa.

The step of holding pressure: The predefined pressure is held for 3-30minutes.

The step of releasing pressure: The pressure of the pressure containeris released to the normal pressure, and the mixture is removed from thecontainer.

Alternatively, in the step of closure, the mixture is first poured intoa packing container which is then airproofed, after that, put thepacking container into the pressure container and close it. Charge themedium for transferring pressure into the pressure container. Thepressure of the pressure container is increased via the medium by apump. After holding for a period of time, release the pressure ofcontainer, and remove the packing container and the mixture therein. Thepacking container may be made of flexible material, such as plastic filmor paper. It also may be made of hard materials, such as metal or glass.The packing container must be pressure transferrable without breakage,osmosis and leakage under super high pressure. Additionally, it does notreact with solvent, raw biomaterials and medium for transferringpressure.

The steps of increasing pressure, holding pressure and releasingpressure can be finished by one step or several steps. If finished byseveral steps, it can be done as the following:

{circle around (1)} To increase the pressure in a ladder-type:increasing pressure in a ladder-type means that pressure is increased tothe first predefined pressure, and hold for certain period time, thenthe pressure is increased to the second predefined higher pressure, andhold certain period time again, after that the pressure is increasedagain, thus repeat until reach the highest predefined pressure, and holdfor certain time, then release the pressure.

{circle around (2)} To increase pressure in a pulse-type: increasingpressure in a pulse-type means that the pressure is increased to thefirst predefined pressure, and hold for certain period time, release thepressure. The pressure is increased again to the second predefinedpressure, hold for certain period time again and release the pressure.Thus repeat several times. The pressure reached each time may be same ornot. The mixture is removed from the container after releasing thepressure at last time.

{circle around (3)} The raw biomaterial may be extracted one time orseveral times, i.e., the raw material which has been extracted undersuper high pressure is mixed with solvent and extracted once again undersuper high pressure. Such extraction can be repeated several times andthe solvent can be the same or not each time.

One or more devices may be placed in the pressure container so that thesuper high pressure can work combined with other processingtechnologies. The devices may be ultrasonator, electric pulser, machinestirrer, heater, or cooler, etc. Such devices can work at any step,several steps or total steps of before increasing pressure, increasingpressure, holding pressure, releasing pressure or after releasingpressure.

The above devices may also be used outside the pressure container beforeextraction. At such circumstance, the processing technologies by use ofthe above devices are called prior treatment.

The extraction solvent of the invention includes water and/or organicsolvent, or the mixture thereof. The organic solvent may includealcohols (for example, methanol, ethanol, isopropyl alcohol, butanol,etc.), ether (for example, dioxane, diethyl ether, petroleum ether,etc.), alkane halide (for example, chloroform, dichloromethane, etc.),ketone (for example, acetone, etc.), hydrocarbon (for example, hexane,industrial solvent oil, etc.), acids (for example, acetic acid, fattyacid etc.), amine (for example, ethanol amine, etc. ), and the mixturethereof.

The medium for transferring pressure of the invention is liquid, whichis the same or not with extraction solvent.

At the step of pretreatment, crashing and formulation, some chemicalsand/or bio-products as auxiliary additives may be added into the mixtureof raw material and solvent. The auxiliary additive added may be one ormore kinds. The auxiliary additives may be act as cosolvent, restrainer,precipitating agent or reactant to improve the performance of extractionunder super high pressure, or to change structures of the extract.

During extraction under super high pressure, the temperature may beincreased or reduced. The medium for transferring pressure and/or themixture of raw material and solvent are heated with heater and cooledwith cooler. The heater and/or cooler are sometimes placed or assembledin the pressure container. Sometimes, the pressure container is put intocooler to cool the mixture and/or medium.

The advantages of the invention are as follows:

A. The extraction solvent has broad scope, including water and/ororganic solvent and the mixture thereof. Therefore, the most propersolvent can be selected as extraction solvent. The present invention hasthe advantage of using much more kinds of solvent over supercritical CO₂extraction.

B. The extraction under high pressure is operated at room temperatureduring which the change of temperature is within 5° C. except of beingheated or cooled. So, the extraction process according to the presentinvention has not the shortages of reaction, denaturation, or loss ofthe active ingredients which appeared in the common heat extraction suchas refluxing, immersion, leaching, boiling and distilling. Thus, it isvery useful for extracting heat sensitive ingredients. Sometimes, theextraction under high pressure is combined with heating or cooling toimprove the performances of extraction, such as increasing the extractyield, changing the ingredients of extract, etc.

C. Theoretically, the solubility of most compounds increases with theincrease of pressure. The pressure of the extraction of the presentinvention is above 100 MPa which is far higher than that of the commonextraction and supercritical CO₂ extraction (usually about 10 MPa).Thus, the solubility of the bioactive ingredients is much better and theextract yield is much higher accordingly.

D. Under super high pressure, the cell membranes are ruptured, and theactive ingredients in the cell can easily enter into the solvent, andthe solvent also can easily enter into the cell. Thus, the extractiontime is greatly shortened. Using the routine methods of extraction, thetime of extraction is much longer. In general, the time of coolimmersion and enzymolysis is from one day to several days; theextraction time of refluxing, boiling or distilling is 6˜8 hour; and theextraction time of supercritical CO₂ is about 2 hour or more. However,the extraction time under super high pressure is generally 2˜15 minutes,and the maximum is no more than 30 minutes.

E. Under super high pressure, the protein and starch are denatured, butnot cleaved, which makes separation and purification more simple andconvenient.

F. The process according to the invention can be used to extract manykinds of active ingredients. The medium for transferring pressure can bethe same or not with the solvent. The pressure equipment is easy to beoperated and controlled. Furthermore, the pressures everywhere of thepressure container are equivalent, which makes the extraction conditionsconstant. The pressure equipment can be used for extracting variouskinds of active ingredients with various kinds of solvents.

G. Because the solubility of ingredients increases under super highpressure, less solvent is needed. The pollutant released is greatlydecreased than other extraction methods.

H. Energy is saved in the process of the invention. One reason is thatthere is no energy consumption at the step of holding pressure.Additionally the compressibility of liquid is less, and the energy tocompress liquid is far less than that to raise temperature and tocompress CO₂ into supercritical state.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the sketch of extracting from the mixture of raw material andsolvent in a packing container under high pressure via medium fortransferring pressure.

FIG. 2 is the sketch of extracting directly from the mixture of rawmaterial and solvent under high pressure.

FIG. 3 is the sketch of extracting from the mixture of raw material andsolvent under high pressure by the movement of piston.

FIG. 4 is the sketch of a pressure container of FIG. 1 containing otherdevices.

FIG. 5 is the sketch of a pressure container of FIG. 2 containing otherdevices.

FIG. 6 is the flow chart of extracting under super high pressure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The details of the process of the invention are further explained withreference to the drawing as following:

FIG. 6, the flow chart of extracting under super high pressure showsthat the procedures of the process of extracting activity ingredient areas the following: The step of pretreatment, crashing and formulation(S1) is that the solid raw biomaterial is pretreated first, includingroguing, cleaning, or macerating, and then is crashed, inflated, orhomogenized, and mixed with solvent by the proper ratio. If theauxiliary additive is required, it can be added during mixing the rawmaterial and solvent. If the prior treatment is required, it shouldworks at this step. Finally, charge the mixture into the packingcontainer and make it airtight. The step of closure (S2) is: put theformulated mixture of step (S1) into the pressure container firstly, andthen close the container. Either way, the mixture is poured into apacking container, which is then airproofed, and finally put the packingcontainer into the pressure container and close it, and after that, themedium for transferring pressure is charged into. The step of increasingpressure (S3) is that the pressure of the pressure container isincreased from normal pressure to the predefined pressure of 100MPa˜1000 MPa at room temperature. The step of holding pressure (S4) isthat the predefined pressure is held for 3-30 minutes. The step ofreleasing pressure (S5) is that the above predefined pressure isreleased to the normal pressure, the container is opened and the mixtureis removed therefrom.

The steps drawn by dotted line in FIG. 6 can be used totally, orpartially, or not used at all according to the raw materials or theactive ingredients.

As shown in FIG. 1, the mixture of raw material and solvent (5) is putinto a packing container (4) which is then airproofed, and then thecontainer (4) is put into the high pressure container (1) which is thenclosed with the end-lid (2). The medium for transferring pressure (6) isintroduced into the container (1) through the connecting tube line (3).The medium (6) is pressed by pump or supercharger to increase thepressure of container (1) to the predefined pressure of 100 MPa˜1000MPa. The predefined pressure is held for 3-30 minutes, and then releasedto normal pressure. The end-lid (2) is removed, and the packingcontainer (4) is taken from the container (1), and then the mixture ofraw material and solvent (5) is removed from the container (4).

As shown in FIG. 2, the mixture of raw material and solvent (5) ischarged into the high pressure container (1) and then the container (1)is closed with the end-lid (2). The pressure of container (1) isincreased to the predefined pressure of 100 MPa˜1000 MPa by pump orsupercharger. After that, the steps of holding pressure and releasingpressure proceed. Alternatively, the pressure container (1) is closedwith lid (2) firstly, and then the mixture (5) is introduced into thecontainer (5) through connecting tube (3) by pump or supercharger. Afterthat, steps of increasing pressure, holding pressure and releasingpressure proceed.

As shown in FIG. 3, the mixture of raw material and solvent (5) ischarged into the pressure container (1), but the end-lid (2) of FIG. 2is replaced by the piston (7). The steps of increasing pressure, holdingpressure and releasing pressure are accomplished by the movement ofpiston (7).

As shown in FIG. 4, the device (8) is placed or assembled in the highpressure container (1) of FIG. 1, wherein the mechanical stirrer is notincluded in the device (8).

As shown in FIG. 5, the device (8) is placed or assembled in the highpressure container (1) of FIG. 2, wherein the mechanical stirrer isincluded.

EXAMPLES Example 1

To extract flavonoids from the gingko leaves:

Take off the impurities in gingkgo leaves, and crash the gingko leavesinto powder with a pulverizer, and then mix 1 g powder with 100 mlwater. The mixture is sealed into a plastic film bag which is then putinto the high pressure container. Close the container, and thenintroduce the mixture of kerosene and transformer oil as medium fortransferring pressure. Increase the pressure of the pressure containerto 500 MPa via the medium by a supercharger. Hold this pressure for 10minutes, and then release it to normal pressure. The container is openedand the mixture is removed from the bag.

The comparing results of extracting under super high pressure and theroutine extraction technologies are shown as following:

The content of Extraction flavonoids No. Formulated mixture technologyin the fluid extract 1 1 g gingko leaves + Super high pressure 3.10mg/ml 100 ml water 500 MPa, 10 minutes 2 1 g gingko leaves + Boiling (1hour) 2.43 mg/ml 100 ml water

Example 2

To extract the tea polyphenols from the tea leaves

Style 2: Take off the impurities in tea leaves and crash tea leaves intopowder. Mix 3 g powder of tea leaves with 540 ml water and 0.5 ml of 75%ethanol as the auxiliary additive. The above mixture is sealed into aplastic film bag which is then put into the pressure container. Closethe container, and then introduce the mixture of kerosene andtransformer oil as medium for transferring pressure. Increase thepressure of the pressure container to 460 MPa via the medium by asupercharger. Hold this pressure for 10 minutes, and then release it tonormal pressure. The container is opened and the mixture is removed fromthe bag.

Style 3: Take off the impurities in tea leaves and crash the tea leavesinto powder. Mix 3 g powder of tea leaves with 360 ml of 80% ethanol.The above mixture is sealed into a plastic film bag which is then putinto the pressure container. Close the container, and then introducewater as the medium for transferring pressure. Increase the pressure ofthe pressure container to 380 MPa via the medium by a supercharger. Holdthis pressure for 10 minutes, and then release it to normal pressure.The container is opened and the mixture is removed from the bag.

The results, obtained by the extraction technology under super highpressure and the routine extraction technologies are shown respectivelyas following:

The content of tea Formulated mixture and polyphenols in No. Extractiontechnology the fluid extract 1 3 g tea leaves + 500 ml water 24.0 mg/mlBoiling, 1 hour 2 3 g tea leaves + 540 ml water 18.68 mg/ml  Super highpressure of 460 MPa, 10 minutes 3 3 g tea leaves + 360 ml 80% 28.0 mg/mlethanol Super high pressure 380 MPa, 10 minutes

Example 3

To Extract Baicalein from Radix Scutellariae

Take off the impurities in Radix Scutellariae; and crash the RadixScutellariae with pulverizer. Mix 5 g powder of Radix Scutellariae with100 ml of 60% methanol. Treat the mixture for 10 minutes with aultrasonator. The above mixture is sealed into a plastic film bag whichis immersed for 4 hours at room temperature. Put the bag into thepressure container, and close the container. Then introduce the mixtureof kerosene and transformer oil as medium for transferring pressure.Increase the pressure of the pressure container to 200 MPa via themedium by a supercharger. Hold this pressure for 5 minutes, and thenrelease it to normal pressure. Increase the pressure of the pressurecontainer to 600 MPa. Hold this pressure for 5 minutes, and then releaseit to normal pressure. The container is opened and the mixture isremoved from the bag.

The results, obtained by the extraction technology under super highpressure and the routine extraction technologies are shown respectivelyas following:

The content of Baicalein in the No. Formulated mixture Extractiontechnology fluid extract 1 5 g Radix Scutellariae + Under super 3.10mg/ml 100 ml methanol high pressure 2 5 g Radix Scutellariae + Heat tillreflux at 1.23 mg/ml 100 ml methanol 100° C., 2.5 h.

INDUSTRIAL APPLICABILITY

The process for extracting small molecular ingredients from biologicalmaterials under super high pressure according to the invention isusually operated at room temperature. Thus, the denatured and loss ofthe active ingredients caused by heating are avoided. A large range ofsolvents can be used in this technology to extract the hydrophilic andlipophilic active ingredients. Additionally, it has advantages such asshorter extraction time, higher extract yield, easier to be separatedand purified. It is a fast extraction technology with high-yield forsmall molecular active ingredients.

1. A process of extracting small molecular ingredients with molecularweight lower than 10,000 from a biological material under super highpressure, comprising the following steps: pretreating the biologicalmaterial by pulverizing and then mixing with a first portion of solventto obtain a first mixture; adding the first mixture into a pressurecontainer; closing the container; increasing pressure in the pressurecontainer from atmospheric pressure to a predefined pressure of 100MPa-1000 Mpa at a temperature within 5° C. of room temperature; holdingthe predefined pressure for 3-30 minutes; releasing pressure in thepressure container; and removing the first mixture from the container;wherein the small molecular ingredients comprise flavonoids extractedfrom ginkgo leaves, polyphenols extracted from tea leaves or baicalein.2. The process according to claim 1, further comprising the steps of:adding the first mixture into a packing container, sealing the packingcontainer and subsequently putting the packing container into thepressure container; adding a medium for transferring pressure into thepressure container; and removing the first mixture from the packingcontainer after releasing pressure in the pressure container.
 3. Theprocess according to claim 2, wherein the medium is liquid.
 4. Theprocess according to claim 1, further comprising the step of increasingpressure in the pressure container to a predefined pressure of 100MPa-1000 MPa for a second time.
 5. The process according to claim 1wherein, the step of increasing pressure in the pressure container isdone in a ladder-type fashion.
 6. The process according to claim 1,wherein the step of increasing pressure in the pressure container isdone in a pulse-type fashion.
 7. The process according to claim 1,wherein the biological material is removed from the first solvent and ismixed with a second portion of a second solvent to obtain a secondmixture that is added into the pressure container and the steps ofincreasing pressure, holding pressure and releasing pressure arerepeated with the second mixture, wherein the first solvent and thesecond solvent can be the same or different.
 8. The process according toclaim 1, wherein the process further comprises other processingtechnologies selected from the group consisting of ultrasonicprocessing, electric pulse processing and mechanical agitation.
 9. Theprocess according to claim 1, wherein said solvent comprises water, anorganic solvent, or the mixture thereof.
 10. The process according toclaim 1, further comprising adding an auxiliary additive into the firstmixture.
 11. The process according to claim 1, further comprising thestep of cooling the pressure container.
 12. The process according toclaim 11, wherein the step of cooling is accomplished by disposing acooler within the pressure container.
 13. The process according to claim11, wherein the step of cooling is accomplished by placing the pressurecontainer into a cooler.
 14. The process according to claim 1, wherein,the step of increasing pressure is performed in steps.
 15. The processaccording to claim 1, wherein, the step of releasing pressure isperformed in steps.
 16. The process according to claim 1, wherein, thestep of increasing pressure is performed at room temperature.